Phylogenetically Distant BABY BOOM Genes From Setaria italica Induce Parthenogenesis in Rice.

The combination of apomixis and hybrid production is hailed as the holy grail of agriculture for the ability of apomixis to fix heterosis of F1 hybrids in succeeding generations, thereby eliminating the need for repeated crosses to produce F1 hybrids. Apomixis, asexual reproduction through seed, achieves this feat by circumventing two processes that are fundamental to sexual reproduction (meiosis and fertilization) and replacing them with apomeiosis and parthenogenesis, resulting in seeds that are clonal to the maternal parent. Parthenogenesis, embryo development without fertilization, has been genetically engineered in rice, maize, and pearl millet using PsASGR-BABY BOOM-like (PsASGR-BBML) transgenes and in rice using the OsBABY BOOM1 (OsBBM1) cDNA sequence when expressed under the control of egg cell-specific promoters. A phylogenetic analysis revealed that BABY BOOM (BBM)/BBML genes from monocots cluster within three different clades. The BBM/BBML genes shown to induce parthenogenesis cluster within clade 1 (the ASGR-BBML clade) along with orthologs from other monocot species, such as Setaria italica. For this study, we tested the parthenogenetic potential of three BBM transgenes from S. italica, each a member of a different phylogenetic BBM clade. All transgenes were genomic constructs under the control of the AtDD45 egg cell-specific promoter. All SiBBM transgenes induced various levels of parthenogenetic embryo development, resulting in viable haploid T1 seedlings. Poor seed set and lower haploid seed production were characteristics of multiple transgenic lines. The results presented in this study illustrate that further functional characterization of BBMs in zygote/embryo development is warranted.

Controlled Induction of Parthenogenesis in Transgenic Rice via Post-translational Activation of PsASGR-BBML.

Modern plant breeding programs rely heavily on the generation of homozygous lines, with the traditional process requiring the inbreeding of a heterozygous cross for five to six generations. Doubled haploid (DH) technology, a process of generating haploid plants from an initial heterozygote, followed by chromosome doubling, reduces the process to two generations. Currently established in vitro methods of haploid induction include androgenesis and gynogenesis, while in vivo methods are based on uni-parental genome elimination. Parthenogenesis, embryogenesis from unfertilized egg cells, presents another potential method of haploid induction. PsASGR-BABY BOOM-like, an AP2 transcription factor, induces parthenogenesis in a natural apomictic species, Pennisetum squamulatum (Cenchrus squamulatus) and PsASGR-BBML transgenes promote parthenogenesis in several crop plants, including rice, maize, and pearl millet. The dominant nature of PsASGR-BBML transgenes impedes their use in DH technology. Using a glucocorticoid-based post-translational regulation system and watering with a 100 µM DEX solution before anthesis, PsASGR-BBML can be regulated at the flowering stage to promote parthenogenesis. Conditional expression presents a novel opportunity to use parthenogenetic genes in DH production technology and to elucidate the molecular mechanism underlying parthenogenetic embryogenesis.

Gene activation via Cre/lox-mediated excision in cowpea (Vigna unguiculata).

KEY MESSAGE: Expression of Cre recombinase by AtRps5apro or AtDD45pro enabled Cre/lox-mediated recombination at an early embryonic developmental stage upon crossing, activating transgenes in the hybrid cowpea and tobacco. Genetic engineering ideally results in precise spatiotemporal control of transgene expression. To activate transgenes exclusively in a hybrid upon fertilization, we evaluated a Cre/lox-mediated gene activation system with the Cre recombinase expressed by either AtRps5a or AtDD45 promoters that showed activity in egg cells and young embryos. In crosses between Cre recombinase lines and transgenic lines harboring a lox-excision reporter cassette with ZsGreen driven by the AtUbq3 promoter after Cre/lox-mediated recombination, we observed complete excision of the lox-flanked intervening DNA sequence between the AtUbq3pro and the ZsGreen coding sequence in F1 progeny upon genotyping but no ZsGreen expression in F1 seeds or seedlings. The incapability to observe ZsGreen fluorescence was attributed to the activity of the AtUbq3pro. Strong ZsGreen expression in F1 seeds was observed after recombination when ZsGreen was driven by the AtUbq10 promoter. Using the AtDD45pro to express Cre resulted in more variation in recombination frequencies between transgenic lines and crosses. Regardless of the promoter used to regulate Cre, mosaic F1 progeny were rare, suggesting gene activation at an early embryo-developmental stage. Observation of ZsGreen-expressing tobacco embryos at the globular stage from crosses with the AtRps5aproCre lines pollinated by the AtUbq3prolox line supported the early activation mode.

Clonal Reproduction through Seeds in Sight for Crops.

Apomixis or asexual reproduction through seeds, enables the preservation of hybrid vigor. Hybrids are heterozygous and segregate for genotype and phenotype upon sexual reproduction. While apomixis, that is, clonal reproduction, is intuitively antithetical to diversity, it is rarely obligate and actually provides a mechanism to recover and maintain superior hybrid gene combinations for which sexual reproduction would reveal deleterious alleles in less fit genotypes. Apomixis, widespread across flowering plant orders, does not occur in major crop species, yet its introduction could add a valuable tool to the breeder's toolbox. In the past decade, discovery of genetic mechanisms regulating meiosis, embryo and endosperm development have facilitated proof-of-concept for the synthesis of apomixis, bringing apomictic crops closer to reality.

Genotypic and phenotypic evaluation of off-type grasses in hybrid Bermudagrass [Cynodon dactylon (L.) Pers. x C. transvaalensis Burtt-Davy] putting greens using genotyping-by-sequencing and morphological characterization.

BACKGROUND: Interspecific hybrid bermudagrass [Cynodon dactylon (L.) Pers. x C. transvaalensis Burtt-Davy] is one of the most widely used grasses on golf courses, with cultivars derived from 'Tifgreen' or 'Tifdwarf' particularly used for putting greens. Many bermudagrass cultivars established for putting greens can be genetically unstable and lead to the occurrence of undesirable off-type grasses that vary in phenotype. The objective of this research was to genetically and phenotypically differentiate off-type grasses and hybrid cultivars. Beginning in 2013, off-type and desirable hybrid bermudagrass samples were collected from golf course putting greens in the southeastern United States and genetically and phenotypically characterized using genotyping-by-sequencing and morphology. RESULTS: Genotyping-by-sequencing determined that 11% (5) of off-type and desirable samples from putting greens were genetically divergent from standard cultivars such as Champion, MiniVerde, Tifdwarf, TifEagle, and Tifgreen. In addition, genotyping-by-sequencing was unable to genetically distinguish all standard cultivars from one another due to their similar origin and clonal propagation; however, over 90,000 potentially informative nucleotide variants were identified among the triploid hybrid cultivars. CONCLUSIONS: Although few genetic differences were found in this research, samples harvested from golf course putting greens had variable morphology and were clustered into three distinct phenotypic groups. The majority of off-type grasses in hybrid bermudagrass putting greens were genetically similar with variable morphological traits. Off-type grasses within golf course putting greens have the potential to compromise putting surface functionality and aesthetics.

Pearl millet genome sequence provides a resource to improve agronomic traits in arid environments.

Pearl millet [Cenchrus americanus (L.) Morrone] is a staple food for more than 90 million farmers in arid and semi-arid regions of sub-Saharan Africa, India and South Asia. We report the ∼1.79 Gb draft whole genome sequence of reference genotype Tift 23D2B1-P1-P5, which contains an estimated 38,579 genes. We highlight the substantial enrichment for wax biosynthesis genes, which may contribute to heat and drought tolerance in this crop. We resequenced and analyzed 994 pearl millet lines, enabling insights into population structure, genetic diversity and domestication. We use these resequencing data to establish marker trait associations for genomic selection, to define heterotic pools, and to predict hybrid performance. We believe that these resources should empower researchers and breeders to improve this important staple crop.

Apomixis: Engineering the Ability to Harness Hybrid Vigor in Crop Plants.

Apomixis, commonly defined as asexual reproduction through seed, is a reproductive trait that occurs in only a few minor crops, but would be highly valuable in major crops. Apomixis results in seed-derived progenies that are genetically identical to their maternal parent. The advantage of apomixis would lie in seed propagation of elite food, feed, and biofuel crops that are heterozygous such as hybrid corn and switchgrass or self-pollinating crops for which no commercial-scale hybrid production system is available. While hybrid plants often outperform parental lines in growth and higher yields, production of hybrid seed is accomplished through carefully controlled, labor intensive crosses. Both small farmers in developing countries who produce their own seed and commercial companies that market hybrid seed could benefit from the establishment of engineered apomixis in plants. In this chapter, we review what has been learned from studying natural apomicts and mutations in sexual plants leading to apomixis-like development, plus discuss how the components of apomixis could be successfully engineered in plants.

Haploid embryo production in rice and maize induced by PsASGR-BBML transgenes.

KEY MESSAGE: The PsASGR - BBML transgene, derived from a wild apomictic grass species, can induce parthenogenesis, embryo formation without fertilization, in rice and maize, leading to the formation of haploid plants. The ability to engineer apomictic crop plants using genes identified from naturally occurring apomicts will depend on the ability of those genes to function in crop plants. The PsASGR-BBML transgene, derived from the apomictic species Pennisetum squamulatum, promotes parthenogenesis in sexual pearl millet, a member of the same genus, leading to the formation of haploid embryos. This study determined that the PsASGR-BBML transgene can induce haploid embryo development in two major monocot crops, maize and rice. Transgene variations tested included two different promoters and the use of both genomic and cDNA PsASGR-BBML-derived sequences. Haploid plants were recovered from mature caryopses (seed) of rice and maize lines at variable rates. The PsASGR-BBML transgenes failed to induce measurable haploid seed development in the model genetic plant system Arabidopsis thaliana. Complexity of embryo development, as documented in transgenic rice lines, identifies the need for further characterization of the PsASGR-BBML gene.

In Silico and Fluorescence In Situ Hybridization Mapping Reveals Collinearity between the Pennisetum squamulatum Apomixis Carrier-Chromosome and Chromosome 2 of Sorghum and Foxtail Millet.

Apomixis, or clonal propagation through seed, is a trait identified within multiple species of the grass family (Poaceae). The genetic locus controlling apomixis in Pennisetum squamulatum (syn Cenchrus squamulatus) and Cenchrus ciliaris (syn Pennisetum ciliare, buffelgrass) is the apospory-specific genomic region (ASGR). Previously, the ASGR was shown to be highly conserved but inverted in marker order between P. squamulatum and C. ciliaris based on fluorescence in situ hybridization (FISH) and varied in both karyotype and position of the ASGR on the ASGR-carrier chromosome among other apomictic Cenchrus/Pennisetum species. Using in silico transcript mapping and verification of physical positions of some of the transcripts via FISH, we discovered that the ASGR-carrier chromosome from P. squamulatum is collinear with chromosome 2 of foxtail millet and sorghum outside of the ASGR. The in silico ordering of the ASGR-carrier chromosome markers, previously unmapped in P. squamulatum, allowed for the identification of a backcross line with structural changes to the P. squamulatum ASGR-carrier chromosome derived from gamma irradiated pollen.

A parthenogenesis gene of apomict origin elicits embryo formation from unfertilized eggs in a sexual plant.

Apomixis is a naturally occurring mode of asexual reproduction in flowering plants that results in seed formation without the involvement of meiosis or fertilization of the egg. Seeds formed on an apomictic plant contain offspring genetically identical to the maternal plant. Apomixis has significant potential for preserving hybrid vigor from one generation to the next in highly productive crop plant genotypes. Apomictic Pennisetum/Cenchrus species, members of the Poaceae (grass) family, reproduce by apospory. Apospory is characterized by apomeiosis, the formation of unreduced embryo sacs derived from nucellar cells of the ovary and, by parthenogenesis, the development of the unreduced egg into an embryo without fertilization. In Pennisetum squamulatum (L.) R.Br., apospory segregates as a single dominant locus, the apospory-specific genomic region (ASGR). In this study, we demonstrate that the PsASGR-BABY BOOM-like (PsASGR-BBML) gene is expressed in egg cells before fertilization and can induce parthenogenesis and the production of haploid offspring in transgenic sexual pearl millet. A reduction of PsASGR-BBML expression in apomictic F1 RNAi transgenic plants results in fewer visible parthenogenetic embryos and a reduction of embryo cell number compared with controls. Our results endorse a key role for PsASGR-BBML in parthenogenesis and a newly discovered role for a member of the BBM-like clade of APETALA 2 transcription factors. Induction of parthenogenesis by PsASGR-BBML will be valuable for installing parthenogenesis to synthesize apomixis in crops and will have further application for haploid induction to rapidly obtain homozygous lines for breeding.

Selection and validation of reference genes for gene expression analysis in apomictic and sexual Cenchrus ciliaris.

BACKGROUND: Apomixis is a naturally occurring asexual mode of seed reproduction resulting in offspring genetically identical to the maternal plant. Identifying differential gene expression patterns between apomictic and sexual plants is valuable to help deconstruct the trait. Quantitative RT-PCR (qRT-PCR) is a popular method for analyzing gene expression. Normalizing gene expression data using proper reference genes which show stable expression under investigated conditions is critical in qRT-PCR analysis. We used qRT-PCR to validate expression and stability of six potential reference genes (EF1alpha, EIF4A, UBCE, GAPDH, ACT2 and TUBA) in vegetative and reproductive tissues of B-2S and B-12-9 accessions of C. ciliaris. FINDINGS: Among tissue types evaluated, EF1alpha showed the highest level of expression while TUBA showed the lowest. When all tissue types were evaluated and compared between genotypes, EIF4A was the most stable reference gene. Gene expression stability for specific ovary stages of B-2S and B-12-9 was also determined. Except for TUBA, all other tested reference genes could be used for any stage-specific ovary tissue normalization, irrespective of the mode of reproduction. CONCLUSION: Our gene expression stability assay using six reference genes, in sexual and apomictic accessions of C. ciliaris, suggests that EIF4A is the most stable gene across all tissue types analyzed. All other tested reference genes, with the exception of TUBA, could be used for gene expression comparison studies between sexual and apomictic ovaries over multiple developmental stages. This reference gene validation data in C. ciliaris will serve as an important base for future apomixis-related transcriptome data validation.

Recombination within the apospory specific genomic region leads to the uncoupling of apomixis components in Cenchrus ciliaris.

Apomixis enables the clonal propagation of maternal genotypes through seed. If apomixis could be harnessed via genetic engineering or introgression, it would have a major economic impact for agricultural crops. In the grass species Pennisetum squamulatum and Cenchrus ciliaris (syn. P. ciliare), apomixis is controlled by a single dominant "locus", the apospory-specific genomic region (ASGR). For P. squamulatum, 18 published sequenced characterized amplified region (SCAR) markers have been identified which always co-segregate with apospory. Six of these markers are conserved SCARs in the closely related species, C. ciliaris and co-segregate with the trait. A screen of progeny from a cross of sexual × apomictic C. ciliaris genotypes identified a plant, A8, retaining two of the six ASGR-linked SCAR markers. Additional and newly identified ASGR-linked markers were generated to help identify the extent of recombination within the ASGR. Based on analysis of missing markers, the A8 recombinant plant has lost a significant portion of the ASGR but continues to form aposporous embryo sacs. Seedlings produced from aposporous embryo sacs are 6× in ploidy level and hence the A8 recombinant does not express parthenogenesis. The recombinant A8 plant represents a step forward in reducing the complexity of the ASGR locus to determine the factor(s) required for aposporous embryo sac formation and documents the separation of expression of the two components of apomixis in C. ciliaris.

Evolution of the apomixis transmitting chromosome in Pennisetum.

BACKGROUND: Apomixis is an intriguing trait in plants that results in maternal clones through seed reproduction. Apomixis is an elusive, but potentially revolutionary, trait for plant breeding and hybrid seed production. Recent studies arguing that apomicts are not evolutionary dead ends have generated further interest in the evolution of asexual flowering plants. RESULTS: In the present study, we investigate karyotypic variation in a single chromosome responsible for transmitting apomixis, the Apospory-Specific Genomic Region carrier chromosome, in relation to species phylogeny in the genera Pennisetum and Cenchrus. A 1 kb region from the 3' end of the ndhF gene and a 900 bp region from trnL-F were sequenced from 12 apomictic and eight sexual species in the genus Pennisetum and allied genus Cenchrus. An 800 bp region from the Apospory-Specific Genomic Region also was sequenced from the 12 apomicts. Molecular cytological analysis was conducted in sixteen Pennisetum and two Cenchrus species. Our results indicate that the Apospory-Specific Genomic Region is shared by all apomictic species while it is absent from all sexual species or cytotypes. Contrary to our previous observations in Pennisetum squamulatum and Cenchrus ciliaris, retrotransposon sequences of the Opie-2-like family were not closely associated with the Apospory-Specific Genomic Region in all apomictic species, suggesting that they may have been accumulated after the Apospory-Specific Genomic Region originated. CONCLUSIONS: Given that phylogenetic analysis merged Cenchrus and newly investigated Pennisetum species into a single clade containing a terminal cluster of Cenchrus apomicts, the presumed monophyletic origin of Cenchrus is supported. The Apospory-Specific Genomic Region likely preceded speciation in Cenchrus and its lateral transfer through hybridization and subsequent chromosome repatterning may have contributed to further speciation in the two genera.

Genetic mapping of the apospory-specific genomic region in Pennisetum squamulatum using retrotransposon-based molecular markers.

Pennisetum squamulatum reproduces by apomixis, a type of asexual reproduction through seeds. Apomixis in P. squamulatum is transmitted as a dominant Mendelian trait, and a genomic region, the apospory-specific genomic region (ASGR), is sufficient for inheritance of the trait. The ASGR is physically large (>50 Mb), highly heterochromatic, hemizygous, and recombinationally suppressed. These characteristics have hindered high-resolution genetic mapping and map-based cloning of apomixis genes. In this study, the long terminal repeat (LTR) regions of ASGR-abundant retrotransposons in the genome of P. squamulatum and ASGR-linked bacterial artificial chromosome clones were identified and sequenced for designing LTR-specific primers. Two hundred and ninety single-dose sequence specific amplified polymorphism (SSAP) markers were generated from 38 primer combinations. The SSAP markers combined with two previous ASGR-mapped markers were used for genetic linkage analysis and construction of a genetic map resulting in the formation of 27 linkage groups at LOD 10, one of which contained >60% of the SSAP markers. After removing identical markers (identical band scoring) on the largest linkage group, 46 markers were finally used for genetic mapping at LOD 10. The markers distributed across 10 different loci covering 19 cM; however, 45 markers were distributed within 9 cM. Six markers were recovered and sequenced. Five markers were successfully converted into sequence characterized amplified regions (SCARs). Segregation of SCAR markers was not always consistent with the SSAP markers of origin suggesting a greater level of error in the SSAP map resulting in an inflated map distance for the ASGR. One SCAR marker (Pst 56-1205-400) detected expression of an ASGR retrotransposon in root, anther, leaf and ovary of P. squamulatum, although sequencing of the RT-PCR product failed to find a functional open reading frame for the transcript.

Sequence analysis of bacterial artificial chromosome clones from the apospory-specific genomic region of Pennisetum and Cenchrus.

Apomixis, asexual reproduction through seed, is widespread among angiosperm families. Gametophytic apomixis in Pennisetum squamulatum and Cenchrus ciliaris is controlled by the apospory-specific genomic region (ASGR), which is highly conserved and macrosyntenic between these species. Thirty-two ASGR bacterial artificial chromosomes (BACs) isolated from both species and one ASGR-recombining BAC from P. squamulatum, which together cover approximately 2.7 Mb of DNA, were used to investigate the genomic structure of this region. Phrap assembly of 4,521 high-quality reads generated 1,341 contiguous sequences (contigs; 730 from the ASGR and 30 from the ASGR-recombining BAC in P. squamulatum, plus 580 from the C. ciliaris ASGR). Contigs containing putative protein-coding regions unrelated to transposable elements were identified based on protein similarity after Basic Local Alignment Search Tool X analysis. These putative coding regions were further analyzed in silico with reference to the rice (Oryza sativa) and sorghum (Sorghum bicolor) genomes using the resources at Gramene (www.gramene.org) and Phytozome (www.phytozome.net) and by hybridization against sorghum BAC filters. The ASGR sequences reveal that the ASGR (1) contains both gene-rich and gene-poor segments, (2) contains several genes that may play a role in apomictic development, (3) has many classes of transposable elements, and (4) does not exhibit large-scale synteny with either rice or sorghum genomes but does contain multiple regions of microsynteny with these species.

Comparative physical mapping of the apospory-specific genomic region in two apomictic grasses: Pennisetum squamulatum and Cenchrus ciliaris.

In gametophytic apomicts of the aposporous type, each cell of the embryo sac is genetically identical to somatic cells of the ovule because they are products of mitosis, not of meiosis. The egg of the aposporous embryo sac follows parthenogenetic development into an embryo; therefore, uniform progeny result even from heterozygous plants, a trait that would be valuable for many crop species. Attempts to introgress apomixis from wild relatives into major crops through traditional breeding have been hindered by low or no recombination within the chromosomal region governing this trait (the apospory-specific genomic region or ASGR). The lack of recombination also has been a major obstacle to positional cloning of key genes. To further delineate and characterize the nonrecombinant ASGR, we have identified eight new ASGR-linked, AFLP-based molecular markers, only one of which showed recombination with the trait for aposporous embryo sac development. Bacterial artificial chromosome (BAC) clones identified with the ASGR-linked AFLPs or previously mapped markers, when mapped by fluorescence in situ hybridization in Pennisetum squamulatum and Cenchrus ciliaris, showed almost complete macrosynteny between the two apomictic grasses throughout the ASGR, although with an inverted order. A BAC identified with the recombinant AFLP marker mapped most proximal to the centromere of the ASGR-carrier chromosome in P. squamulatum but was not located on the ASGR-carrier chromosome in C. ciliaris. Exceptional regions where synteny was disrupted probably are nonessential for expression of the aposporous trait. The ASGR appears to be maintained as a haplotype even though its position in the genome can be variable.

A segment of the apospory-specific genomic region is highly microsyntenic not only between the apomicts Pennisetum squamulatum and buffelgrass, but also with a rice chromosome 11 centromeric-proximal genomic region.

Bacterial artificial chromosome (BAC) clones from apomicts Pennisetum squamulatum and buffelgrass (Cenchrus ciliaris), isolated with the apospory-specific genomic region (ASGR) marker ugt197, were assembled into contigs that were extended by chromosome walking. Gene-like sequences from contigs were identified by shotgun sequencing and BLAST searches, and used to isolate orthologous rice contigs. Additional gene-like sequences in the apomicts' contigs were identified by bioinformatics using fully sequenced BACs from orthologous rice contigs as templates, as well as by interspecies, whole-contig cross-hybridizations. Hierarchical contig orthology was rapidly assessed by constructing detailed long-range contig molecular maps showing the distribution of gene-like sequences and markers, and searching for microsyntenic patterns of sequence identity and spatial distribution within and across species contigs. We found microsynteny between P. squamulatum and buffelgrass contigs. Importantly, this approach also enabled us to isolate from within the rice (Oryza sativa) genome contig Rice A, which shows the highest microsynteny and is most orthologous to the ugt197-containing C1C buffelgrass contig. Contig Rice A belongs to the rice genome database contig 77 (according to the current September 12, 2003, rice fingerprint contig build) that maps proximal to the chromosome 11 centromere, a feature that interestingly correlates with the mapping of ASGR-linked BACs proximal to the centromere or centromere-like sequences. Thus, relatedness between these two orthologous contigs is supported both by their molecular microstructure and by their centromeric-proximal location. Our discoveries promote the use of a microsynteny-based positional-cloning approach using the rice genome as a template to aid in constructing the ASGR toward the isolation of genes underlying apospory.

High-resolution physical mapping in Pennisetum squamulatum reveals extensive chromosomal heteromorphism of the genomic region associated with apomixis.

Gametophytic apomixis is asexual reproduction as a consequence of parthenogenetic development of a chromosomally unreduced egg. The trait leads to the production of embryos with a maternal genotype, i.e. progeny are clones of the maternal plant. The application of the trait in agriculture could be a tremendous tool for crop improvement through conventional and nonconventional breeding methods. Unfortunately, there are no major crops that reproduce by apomixis, and interspecific hybridization with wild relatives has not yet resulted in commercially viable germplasm. Pennisetum squamulatum is an aposporous apomict from which the gene(s) for apomixis has been transferred to sexual pearl millet by backcrossing. Twelve molecular markers that are linked with apomixis coexist in a tight linkage block called the apospory-specific genomic region (ASGR), and several of these markers have been shown to be hemizygous in the polyploid genome of P. squamulatum. High resolution genetic mapping of these markers has not been possible because of low recombination in this region of the genome. We now show the physical arrangement of bacterial artificial chromosomes containing apomixis-linked molecular markers by high resolution fluorescence in situ hybridization on pachytene chromosomes. The size of the ASGR, currently defined as the entire hemizygous region that hybridizes with apomixis-linked bacterial artificial chromosomes, was estimated on pachytene and mitotic chromosomes to be approximately 50 Mbp (a quarter of the chromosome). The ASGR includes highly repetitive sequences from an Opie-2-like retrotransposon family that are particularly abundant in this region of the genome.

Delineation by fluorescence in situ hybridization of a single hemizygous chromosomal region associated with aposporous embryo sac formation in Pennisetum squamulatum and Cenchrus ciliaris.

Apomixis is a means of asexual reproduction by which plants produce embryos without meiosis and fertilization; thus the embryo is of clonal, maternal origin. We previously reported molecular markers showing no recombination with the trait for aposporous embryo sac development in Pennisetum squamulatum and Cenchrus ciliaris, and the collective single-dose alleles defined an apospory-specific genomic region (ASGR). Fluorescence in situ hybridization (FISH) was used to confirm that the ASGR is a hemizygous genomic region and to determine its chromosomal position with respect to rDNA loci and centromere repeats. We also documented chromosome transmission from P. squamulatum in several backcrosses (BCs) with P. glaucum using genomic in situ hybridization (GISH). One to three complete P. squamulatum chromosomes were detected in BC(6), but only one of the three hybridized with the ASGR-linked markers. In P. squamulatum and in all BCs examined, the apospory-linked markers were located in the distal region of the short arm of a single chromosome. All alien chromosomes behaved as univalents during meiosis and segregated randomly in BC(3) and later BC generations, but presence of the ASGR-carrier chromosome alone was sufficient to confer apospory. FISH results support our hypotheses that hemizygosity, proximity to centromeric sequences, and chromosome structure may all play a role in low recombination in the ASGR.